Investigations over the past 5 years have led to the identification of regions of myosin Vb (Myo5B) responsible for its interactions with multiple Rab proteins and other effectors. Our studies have determined distinct regions in myosin Vb that are responsible for binding to Rab11 family members (Rab11a, Rab11b and Rab25), Rab8a and Rab10. Recently, work from patients in Europe and from the Navajo tribe has identified mutations in Myo5B as the cause of the rare congenital diarrhea syndrome Microvillous Inclusion Disease (MVID). Children with MVID have intractable diarrhea due to an apparent loss of absorptive capacity in the small intestine. Morphologically, the small intestinal cells show loss of the apical brush border and the presence of intracellular inclusions that appear to represent intracellular apical membrane vacuoles. All of the patients from the Navajo tribe have a single point mutation that inactivates proper motor function. These findings suggest that mutations in Myo5B lead to alterations in proper trafficking of proteins to the apical membrane and the assembly of the apical brush border. Given our experience with dissecting out the effects of Myo5B mutations on trafficking, we now propose to evaluate the underlying abnormalities that result from loss of function Myo5B mutations in MVID. We hypothesize that Myo5B, in concert with Rab11a and Rab8a, is responsible for the establishment and maintenance of polarized apical membrane structures including the intestinal microvilli. To address this hypothesis, we will first establish an in vitro model of MVID through the reduction of endogenous Myo5B expression and re-expression of Myo5B missense point mutations in polarized CaCo-2 cells in culture. These studies will allow putative recapitulation of the disease phenotype in vitro by re-expression of the Myo5B Navajo mutation in cells with endogenous knockdown. This in vitro cell model can be evaluated for changes in microvillar assembly as well as alterations in cell polarity. In addition, we will be able to manipulate the phenotype in knockdown cells to evaluate particular Rab-associated pathways associated with Myo5B. Finally we will be able to compare observations in the in vitro system with alterations in tissues from patients with the Myo5B Navajo mutation. Second, we will examine the effects of loss of Myo5B in Myo5B knockout mice. By developing both constitutive and intestinal mucosal-targeted deletion models for Myo5B, we will be able to evaluate the effects of Myo5B inactivation on intestinal cell apical polarity and function. These effects will be compared with both in vitro models and the in vivo effects observed in patients with the Navajo mutation. Overall these studies will not only provide information on how mutations in Myo5B can lead to the sequelae of MVID, but will also determine how alterations in Myo5B- regulated trafficking can lead to alterations in intestinal cell polarity. PUBLIC HEALTH RELEVANCE: The intestinal microvilli are required for absorption of fluid and digestion of food. Loss of these structures leads to diarrhea in microvillous inclusion disease and in other maladies such as Celiac disease. This study seeks to understand the basic mechanisms that support the establishment and maintenance of these critical structures.